The kidney is generally recognized as the major production site of erythropoietin (Epo), the physiologic regulator of red cell production. Experimental hypoxia produces a sharp increase in kidney Epo mRNA levels and serum Epo. The cellular oxygen sensing mechanism, the Epo specific transcriptional activating factors, and the genomic sequences necessary for Epo regulation have not been elucidated. In 1986, we isolated two cell lines which inappropriately synthesized Epo. To explain the abnormal Epo transcription and gain new information concerning Epo regulation, we will investigate the following three hypotheses: 1). Genomic alterations removed negative regulatory sequences or inserted positive regulatory sequences; 2) Epo mRNA stability has increased, and 3) putative Epo transcriptional activating factors have been inappropriately expressed. We have explored the first model by restriction mapping and have discovered genomic alterations in the Epo genes from both cells lines. In one case, the alterations are upstream of Epo coding sequences. Current cloning and sequencing experiments are expected to reveal the precise nature of the Epo rearrangements. In a second project we are attempting to identify normal cells which adjust Epo transcription in response to changes in oxygen concentration in vitro. We will assess Epo inducibility in several new human kidney cell lines recently isolated at NIH.